Entropy-Driven Amino Acid-Based Coacervates with Enzyme-Free Metabolism and Prebiotic Robustness

Abstract

Protocells capable of nonenzymatic metabolism and environmental adaptation are essential models for understanding the emergence of cellular life. However, existing protocell designs often lack the robustness or prebiotic relevance to explain how functional supramolecular assemblies could have formed under early Earth conditions. In this study, we demonstrate that simple amino acid derivatives, observed on extraterrestrial bodies and under simulated prebiotic Earth conditions, undergo entropy-driven liquid–liquid phase separation to form membraneless protocells through a self-coacervation process. The synergistic effect of selective enrichment of metabolites and interfacial acceleration in these coacervate microdroplets enhances enzyme-free reactions, including sulfur metabolism and prebiotic pigment synthesis. The protocells are stabilized by water-mediated hydrogen-bonding networks and exhibit exceptional resilience to prebiotically plausible stressorssuch as high salinity (up to 4.0 M NaCl), high concentrations of divalent cations (4.0 M Mg²⁺/Ca²⁺), UV radiation, and extreme temperature fluctuationswhich typically disrupt existing vesicle-based systems. Remarkably, these structures autonomously generate and maintain a proton gradient (ΔpH ≈ 0.6–2.1) across their interfaces, enabling primitive chemiosmotic coupling via Na⁺–H⁺ antiport activity. They also adaptively remodel into compact spherical morphologies in response to sudden environmental changes, thereby preserving structural integrity. By integrating compartmentalization, nonenzymatic catalysis, energy transduction, and stress tolerance within a minimalist amino acid framework, our results establish a geochemically plausible pathway for the formation and persistence of functional protocells. This work highlights the potential of coacervate-based microcompartments to bridge nonliving and living systems by sustaining biochemical complexity under prebiotic conditions.

Introduction

The emergence of protocells capable of compartmentalization, metabolism, and environmental adaptation represents a crucial step in the origin of life. While lipid vesicles, fatty acids, and peptide assemblies have been extensively studied as protocell models, − their reliance on complex molecular architectures and limited stability under prebiotic conditions pose fundamental challenges to their plausibility. , The early Earth was characterized by extreme environmental stressors, including intense UV radiation, high salinity, fluctuating pH, and elevated divalent metal ion concentrations , conditions that disrupt most membrane-based compartments and impede the maintenance of metabolic functions. Furthermore, few existing systems demonstrate the capacity for energy transduction, a central feature of living cells, highlighting a critical gap between rudimentary compartmentalization and functionally active protocells.

Liquid–liquid phase separation (LLPS) offers an alternative pathway for forming membraneless protocells through coacervation. − Such droplets exhibit dynamic responsiveness to environmental changes, − akin to modern biomolecular condensates, , and provide a microcompartmentalization strategy that may have been important under prebiotic conditions. Amino acids, which are prebiotically plausible and have been identified in meteorites and simulated hydrothermal vent systems, − represent attractive building blocks for coacervate formation. However, unmodified amino acids predominantly undergo crystallization rather than coacervation. Inspired by the “sticker-spacer” motifs of intrinsically disordered proteins that drive phase separation in biological systems, we designed simple amino acid derivatives featuring N-terminal hydrophobic caps (e.g., phenyl, tert-butyl) as well as hydrogen-bond-forming motifs, interconnected by flexible linkers. Beyond their role in phase behavior, these hydrophobic capping groups are prebiotically plausible: not only have amino acid N-terminal carbonates and carbamates been shown to form under realistic prebiotic settings, , but small aromatic and alkyl hydrocarbonspotential precursors to such modifiershave also been identified in carbonaceous meteorites and simulated prebiotic mixtures. Thus, this molecular design provides a conceptual bridge between the phase-separating motifs of modern proteins and a chemically plausible prebiotic scenario.

Here, we report the design, characterization, and functional evaluation of self-coacervating protocells based on prebiotically plausible amino acids (SCPAAs). These droplets form via an entropy-driven dehydration mechanism stabilized by water-mediated hydrogen bonding and hydrophobic interactions. We demonstrate that SCPAAs sequester diverse metabolites and metal ions, promote enzyme-free reactions, including sulfur metabolism and prebiotic pigment synthesis, and exhibit unprecedented resistance to prebiotically relevant stressorssuch as high ionic strength (4.0 M NaCl), high concentrations of divalent cations (4.0 M Mg²⁺/Ca²⁺), UV radiation, and extreme temperature fluctuationsall of which are challenges that disrupt traditional protocell models. Notably, SCPAAs spontaneously generate and maintain a proton gradient across their interfaces, enabling primitive chemiosmotic coupling via Na⁺-H⁺ antiport activity. They also adaptively remodel their morphology in response to sudden changes in environmental conditions, thereby preserving structural and functional integrity. By integrating compartmentalization, nonenzymatic catalysis, energy transduction, and stress resilience into a simple amino acid–based system, SCPAAs provide a geochemically realistic and robust framework for protocell formation on the early Earth. This work establishes a new platform for studying protometabolism and underscores the role of phase-separating amino acid assemblies in the origins of cellular life.

Results and Discussion

Design, Fabrication, and Characterization of SCPAA

Biomolecular condensates formed by intrinsically disordered proteins are often described through a stickers-spacers framework (Figure a), where folded binding motifs (stickers) are interconnected by flexible regions (spacers). Drawing inspiration from this, we designed a library of prebiotically relevant amino acids (PAAs) by incorporating hydrophobic capping groups and hydrogen-bonding motifs as rigid stickers, joined by flexible segments to construct a minimal coacervate-based protocell model (Figure a). To emulate spacer flexibility, methylene groups (−CH₂−) were introduced as minimal linkers, enabling rotational freedom essential for dynamic rearrangement.

1.

Design, preparation, and characterization of SCPAAs. (a) Schematic illustration showing the design of PAA inspired by the intrinsically disordered proteins and their ability to form coacervate protocell under the mimic prebiotic scenarios. (b) Pie chart illustrating the probability distribution of coacervate formation compared to those of gel, crystal, and solution based on our designed PAA library. (c) Dynamic light scattering intensity during the cooling process of Z-Pro solution (7.5 mg mL^–1). Inserted vials and Bright field images indicate the transition from homogeneous solution (right) to turbid coacervate dispersion (left); scale bar: 50 μm. (d) Coalescence events observed suggesting the liquid nature of Z-Pro droplet; scale bar: 20 μm. (e, f) Fluorescence recovery after photobleaching (e) and FRAP recovery curves (f) of the coacervates microdroplets; scale bar: 5 μm. Red emission is from nile red. (g) Cryo-SEM image showing the smooth surface of Z-Pro coacervate droplet (10 mg mL^–1); scale bar: 500 nm. (h) TEM image illustrating the Z-Pro coacervate droplet without textured internal structure (20 mg mL^–1); scale bar: 1 μm. (i) Macroscopic semitransparent coacervates (200 mg mL^–1) consisting of transparent section in the bottom and turbid phase in the upper layer. Aging of the turbid coacervates results in the formation of transparent coacervate, accompanied by a transition from the heterogeneous to homogeneous.

Coacervate formation requires balanced intermolecular interactions between PAA–PAA and PAA-water. Unmodified amino acids tend to crystallize due to strong hydrogen-bonding networks. To circumvent this, we introduced N-terminal carbonyl modifications, creating N-carbonylated amino acids (Figure S1). These modifications enhance interactions with water through additional hydrogen-bonding sites while improving molecular packing as effective stickers. Hydrophobic interactions, critical in biomolecular condensates, were further leveraged by incorporating aromatic (phenyl) or aliphatic (tert-butyl) motifs as complementary stickers. This dual strategy yielded carbobenzyloxy- (Z-) and t-butyloxycarbonyl- (Boc-) modified amino acids, which combine hydrogen-bonding capacity with hydrophobicity to stabilize coacervate assembly (Figure S1).

Using our PAA-based compound library, we constructed coacervate protocells via a heating–cooling cycle in ultrapure watera process that facilitates complete dissolution without decomposition at elevated temperatures (Figure S2), promotes homogeneous solution formation, and subsequently enables spontaneous coacervation upon cooling. This approach combines cyclical temperature changes with a self-stabilized acidic milieu (pH < 4) arising from PAA dissolution in pure water, thereby effectively mimicking the fluctuating thermal and mildly acidic conditions of plausible prebiotic environments such as hydrothermal pools (Figure a). The resulting assemblies included coacervates, hydrogels, crystals, and solutions (Figures S3, S4, Tables S1, and S2). Notably, most PAAs initially formed coacervates (Table S1), with half stabilizing into persistent coacervates (Figure b). A majority of these coacervates were derived from prebiotically abundant amino acids, supporting a feasible pathway for protocell emergence through amino acid self-coacervation. Compared to prior protocell models reliant on nucleoside phosphates, oligonucleotides, or peptides, ,, our SCPAA system represents a minimalist advance, enabling exploration of early chemical evolution in prebiotic contexts.

The formation mechanism is exemplified by Z-Pro coacervates. Heating Z-Pro in water produced a homogeneous solution, which upon cooling formed turbid SCPAA droplets (5–20 μm diameter; Videos S1 and S2). Light scattering intensity increased during phase separation (Figure c), and droplet coalescence confirmed the occurrence of liquid–liquid phase separation (Figures d, S5, and Video S3). By tuning the competition and balance of intermolecular interactions via PAA concentration, the critical temperature for coacervate formation and colloidal stability can be readily modulated (Figure S6). Fluorescence recovery after photobleaching (FRAP) assays revealed rapid molecular mobility within Z-Pro coacervates, with >96% fluorescence recovery within 39 s (Figure e,f), indicating fluid, adaptive interiors conducive to environmental responsiveness. Cryogenic scanning electron microscopy (Cryo-SEM) and transmission electron microscopy (TEM) imaging showed smooth surfaces and homogeneous interiors (Figure g, h), contrasting sharply with the bicontinuous sponge or wormlike micelle structures of surfactant- or lipid-based coacervates. − This structural simplicitycoupled with spontaneous assembly and dynamic behavioraligns with early Earth’s low-complexity chemistry, positioning SCPAA as a plausible primitive protocell.

Aging triggered a transition from turbid dispersions to macroscopically phase-separated systems (Figure i). Initially, ellipsoidal coacervates contained coexisting transparent and turbid regions (the latter comprising microscale coacervates). Over time, these coalesced into a single dense, transparent phase (Video S4), mirroring ancient oceanic turbidity shifts caused by colloidal aggregation. − Remarkably, aged coacervates remained stable for >2 years without crystallization (Figure S7), underscoring their robustness as protocell models.

Formation and Stabilization Mechanism of SCPAA

Having established the coacervate-forming capability of single-component PAAs, we next explored the physicochemical principles governing their self-assembly. Coacervate formation depends on the interplay between PAA–PAA and PAA-water interactions. To quantify these effects, we analyzed melting points (reflecting intermolecular interaction) and octanol–water partition coefficients (log P, reflecting hydrophobicity) across the PAA library. Z-modified PAAs forming coacervates exhibited melting points below 120 °C and log P values of 0.9–3.5 (Figure a, Table S1). Similarly, Boc-modified PAAs with coacervate-forming ability showed melting points below 130 °C and log P values of 0.25–3 (Figure S8, Table S2). Outside these ranges, crystallization or dissolution dominated, indicating that weak-to-moderate intermolecular interactions and balanced amphiphilicity are critical for coacervate stability: they prevent excessive aggregation while retaining sufficient aqueous compatibility. This principle was further corroborated by control experiments using amino acids modified with N-benzoyl or N-acetyl groups, in which strong intermolecular interactions or high hydrophilicity led to crystallization or dissolution, respectively (Figures S9, S10, and Table S3).

2.

Formation and evolution mechanism of prebiotically amino acid-based coacervates. (a) Melting point as a function of log P illustrating the condition matrix for coacervates, gels, crystals, and solutions. (b) Bright field microscopic images showing the responsiveness of Z-Pro coacervates toward additives with different concentrations. Scale bars: 20 μm. (c) Van’t Hoff plot showing the temperature dependence of the equilibrium constant (correlation coefficient of 0.995). The standard enthalpy change (ΔH) and standard entropy change (ΔS) for the formation of coacervate were determined using eq 1 in methods: ΔH= 11.11 kJ mol^–1 and ΔS = 75.46 J mol^–1 K^–1. (d) Water content corresponding to coacervate (40 mg mL^–1) with varied aging times. The gradual decrease in water content suggests the occurrence of dehydration. (e, f) Raman spectra of Z-Pro coacervates, with comparison to Z-Pro crystals. (g) Molecular packing modes obtained from AAMD simulation, demonstrating the hydrophobic interaction between Z-Pro molecules and hydrogen-bonding networks among Z-Pro and water.

To elucidate the driving forces underlying coacervation, we titrated Z-Pro coacervates with 1,6-hexanediol (hydrophobic disruptor) and urea (hydrogen-bond disruptor). Increasing concentrations of both agents progressively dissolved coacervates (Figure b). Notably, 1,6-hexanediol induced complete dissolution (turbid → clear transition), whereas urea left residual droplets, collectively indicating that hydrophobic interactions constitute the primary driver of coacervation, with hydrogen bonding playing a secondary role. Temperature-dependent equilibrium analysis using the van’t Hoff equation revealed that coacervation is entirely entropy-driven (ΔS = +75.46 J mol^–1 K^–1), overcoming an enthalpic penalty (ΔH = +11.11 kJ mol^–1) (Figure c, Table S4). The favorable entropy gain arises from the release of water molecules originally bound to Z-Pro into the bulk phase, , as corroborated by the progressive dehydration observed during coacervate aging (Figures d and S11), thereby increasing the overall disorder of the system. The accompanying enthalpic penalty reflects partial disruption of hydrogen bonds. Thus, dehydration-induced entropy gain serves as the principal molecular driving force for coacervation. These findings are consistent with Oparin’s primitive coacervate model, and demonstrate that simple hydrophobic forcesrather than complex multivalent interactions suffice to drive protocell assembly, a mechanism congruent with the limited chemical complexity expected on early Earth.

Raman spectroscopy was employed to probe the intermolecular interactions stabilizing SCPAA. Broadening of C–H stretching vibration peaks (2850–3180 cm^–1) in Z-Pro coacervates, compared to sharp peaks in Z-Pro crystals (Figure e), suggests enhanced hydrophobic interactions in the coacervate phase. In crystalline Z-Pro, distinct peaks at 1646 cm^–1 (CO in benzyloxycarbonyl), 1725 cm^–1 (carboxyl CO), and 3196 cm^–1 (O–H) were observed (Figure f). Upon coacervation, these features shifted: new broad bands emerged at 3360–3570 cm^–1 (O–H/N–H stretching) and 1630–1770 cm^–1 (CO/C–N vibrations), while blue-shifted peaks at 3204 and 3213 cm^–1 indicated reorganization of hydrogen-bonding networks involving water. All-atom molecular dynamics (AAMD) simulations corroborated these findings, revealing dynamic water-mediated hydrogen bonds (Figure g). Notably, a shoulder peak at 3111 cm^–1 and a strong band at 2885 cm^–1 (Figure S12) confirmed the presence of carboxyl-bound water. Together, these interactions form a dynamic noncovalent network that traps molecular organization in a metastable state, suppressing crystallization and enabling SCPAA formation.

Compartmentalization-Enhanced Nonenzymatic Metabolic Reactions

Having established SCPAAs as feasible protocellular coacervates, we next evaluated their capacity to enhance prebiotic metabolic processes. In the open environment on primordial Earth, prebiotic metabolites are prone to diffusion-driven dilution, limiting reaction efficiency and selectivity. , Compartmentalization within protocells could overcome this barrier by concentrating reactants and creating distinct chemical microenvironments, a prerequisite for viable reaction rates and pathway specificity in early life. , We therefore assessed the sequestration capability of SCPAAs using a range of guest molecules, including charged and neutral hydrophobic dyes, prebiotic biomolecules, and metal ions (Figure a–f).

3.

Compartmentalization-enhanced nonenzymatic metabolic reaction. (a–h) Partitioning of different types of components including nile red (a), methyl green (b), indo green (c), glycine (d), FITC-OVA (e), Na⁺ (f) Mg²⁺ (g), and Ca²⁺ (h) in coacervates. The pronounced color in (d) arising from characteristic ninhydrin reaction for amino acid detection. The emergence of macroscopic SCPAA and substantial coacervates droplets in (f–h) suggest the tolerance of SCPAA to high concentration of metal ions. (i) Microscopy images showing SCPAA-accelerated oxidation of cysteine to cystine. Scale bars: 100 μm (top left, bottom left, and bottom right images); 50 μm (top right image). (j) Raman spectra detecting cysteine formation via cysteine oxidation. Characteristic disulfide bond peaks appear in the cysteine system with SCPAA, confirming oxidation-produced cystine as the insoluble material. (k) UV–vis spectra of Z-Tyr coacervates pre- and after-oxidation to melanin-like derivatives. Inset: corresponding photographic images. (l) Fluorescence recovery after photobleaching (FRAP) images of methyl green-loaded Z-Tyr (top) and Z-Tyr_mel (bottom) coacervate droplets. Scale bar = 10 μm.

Confocal microscopy revealed uniform, intense fluorescence within SCPAA droplets when incubated with aromatic dyesneutral (Nile Red), cationic (methyl green), or anionic (indocyanine green)confirming their selective enrichment (Figure a–c). Prebiotic amino acids were similarly sequestered, as evidenced by the dark blue coloration of coacervates (Figure d). This broad uptake arises from the amphiphilic nature of SCPAA, which provides coexisting hydrophilic and hydrophobic microdomains. In contrast, hydrophilic macromolecules like FITC-labeled ovalbumin (OVA) localized preferentially at the coacervate interface rather than the interior (Figure e), indicating molecular weight-dependent selectivity governed by diffusion kinetics, steric exclusion, and electrostatic/hydrophobic interactions.

Notably, SCPAA exhibits exceptional tolerance to high-salinity environments (NaCl) and divalent cations (Mg²⁺/Ca²⁺), withstanding concentrations of at least 4.0 M (Figures f–h, S13, and S14)conditions prevalent in ancient oceans and hydrothermal spring systems. This robustness stands in sharp contrast to fatty acid–based protocells, which undergo crystallization even at mM concentrations of divalent cations, ,, thereby undermining their prebiotic plausibility. The stability of SCPAAs under such extremes positions them as compelling models for early Earth environments. Notably, this high salt tolerance coexists with low internal enrichment of divalent cations (Mg²⁺: 13.3 mM; Ca²⁺: 77.4 mM), indicating that stabilization does not result from ion enrichment, but rather from metal coordination-enhanced hydrophobic interactions at the coacervate interface, which collectively reinforce the coacervate structure (Figures S15–S17).

Furthermore, SCPAA’s selective partitioning behavior could have functioned as a physicochemical sieve, concentrating essential substances within primitive compartments while excluding larger solutes. This may have enabled the synthesis of biomacromolecules and the emergence of rudimentary metabolic pathwayscritical steps in transitioning from nonliving to living systems within SCPAA.

To evaluate SCPAA’s metabolic potential, we tested its ability to accelerate a nonenzymatic reaction analogous to early Earth redox metabolism. Such metabolism underpinned primordial chemical networks, exemplified by prebiotic sulfur reactions that provided catalytic interfaces, electron-transfer mediators, and organic precursors. Crucially, it served as both an engine for energy conversion and a stepping stone for life’s emergence. Sulfur-containing amino acids (e.g., cysteine) detected in carbonaceous chondrites likely formed via prebiotic Strecker-like reactions, supporting the plausibility of this pathway.

We thus selected cysteine-to-cystine oxidation as a simple model to assess nonenzymatic reaction rates. In the presence of SCPAA, 10.0 mM cysteine was oxidized to cystine within 40 min at room temperature, as confirmed by ESI-MS (m/z 261.10) with a yield of 88.9% (Figures S18 and S19). Notably, the coacervate system promoted the formation of insoluble aggregates exhibiting a disulfide bond vibration at 484 cm^–1 and hexagonal crystallinity, consistent with crystalline cysteine (Figures i,j, S20, and Video S5). By contrast, control experiments in aqueous solution without coacervates showed significantly lower yields (14.4%) under identical conditions and produced no such aggregates (Figures i, S19, and Video S6). Together, these results clearly demonstrate that SCPAAs enhance the oxidation of cysteine to cystine.

To determine the origin of this enhancement, we quantified molecular distribution between phases. The cysteine concentration was lower in the coacervates (∼6.0 mM) than in the supernatant (∼10.6 mM), ruling out reactant enrichment as the primary cause (Figure S21). Instead, the distinct hydrophobicity of the reactant and product drives the reaction: cysteine (log P = 0.23) favors the supernatant, while the more hydrophobic cystine (log P = 0.59)similar to Boc-Pro (log P = 0.56)preferentially partitions into the coacervate phase. This spontaneous product removal shifts the reaction equilibrium forward, while the coacervate–supernatant interface provides a catalytically active microenvironment. Together, thermodynamic pulling and interfacial catalysis act synergistically to enhance reaction kinetics.

The resulting disulfide bonds, being covalent, are more robust than noncovalent interactionsa critical feature for maintaining biomolecular integrity under early Earth extremes such as high temperature, UV radiation, and dehydration. The cysteine-to-cystine reaction illustrates how SCPAA interface can amplify reaction efficiency, enabling protometabolic networks to emerge in dilute prebiotic environments. Moreover, the persistence of sulfur-dependent pathways in modern metabolism underscores sulfur’s role as a “chemical relic” of life’s origin. Our findings suggest that SCPAAs could have served as rudimentary metabolic scaffolds, where compartmentalization drives efficient nonenzymatic reactionsa key evolutionary step toward the emergence of protometabolism.

Beyond sulfur-based redox chemistry, this compartmentalization-enhanced protometabolism also facilitates the synthesis of prebiotic pigments. Z-Tyr self-coacervates encapsulating Fe³⁺ exhibit a broadband visible absorption and significantly enhanced UV absorbance compared to pristine Tyr self-coacervates (Figure k), resembling typical melanin absorption. Molecular characterization via ESI-MS spectra confirms the emergence of a melanin-like motif (Figure S22), indicating melanin-like derivatives (Z-Tyr_mel). Strikingly, kinetic analysis revealed rapid compartmentalization-enhanced formation: a detectable signal emerged within 10 min and peaked at 60 min in the coacervate phase, whereas the supernatant required 1 h for initial detection and 6 h to reach its maximum (Figure S23). These results clearly illustrate that SCPAAs enhance prebiotic pigment synthesis through their molecular building blocks.

Notably, these melanin-like derivatives substantially improve coacervate colloidal stability. Fluorescence recovery after photobleaching (FRAP) assays quantitatively showed that Z-Tyr_mel coacervates exhibit markedly slower and reduced recovery relative to Z-Tyr, indicating restricted molecular mobility (Figures l and S24). This finding underpins their observed coalescence resistance and sustained morphological integrity after oxidation (Figures S25 and S26). The persistence of such oxidized coacervates implies that primordial compartments could maintain structural stability while facilitating redox reactionsa prerequisite for sustained proto-metabolic activity.

Taken together, the formation of cystine crystals within SCPAAs and melanin-like aggregates in coacervates jointly illustrate how redox-active molecules, operating within compartmentalized microenvironments, could have driven the transition from abiotic chemistry toward functional protometabolic networks. Rooted in early Earth conditionssuch as hydrothermal activity and mineral catalysisthese processes provide critical insights into the chemistry-to-biology transition at life’s origins.

Autonomous Proton Gradients Generation

While the selective partitioning of metabolites establishes SCPAAs as efficient microreactors, we hypothesized that their prebiotic relevance might extend beyond passive enrichment to include active, energy-transducing functions. Remarkably, these coacervate protocells autonomously generate and sustain a substantial proton gradient between the coacervate and supernatant phases, with an internal pH of 0.71–1.59 sharply offset from the bulk phase (2.09–3.23), yielding a ΔpH of 0.60–2.13 in an amino acid-dependent manner, which was consistently reproduced across independent preparations (n = 3, mean ± SD), as shown by the small error bars in Figure a. Time-resolved monitoring on the representative Z-Pro coacervates further revealed that the ΔpH initially rises from ≈1.39 to ≈1.56 within 24–48 h and then stabilizes at ≈1.30 over 2 weeks (Figure S27), indicating a transient ion-exchange phase followed by a persistent steady state. Collectively, these findings demonstrate that SCPAAs sustain a reproducible, long-lived, and energetically significant proton gradient, thereby endowing prebiotic coacervates with a primitive chemiosmotic-like capacity.

4.

Autonomous proton gradient generation and dynamic exchange in SCPAAs. (a) Differences in pH (ΔpH) between the coacervate and supernatant phases for various Z-amino acids, demonstrating amino acid-dependent proton gradient formation. Data are presented as mean ± SD (n = 3 independent experiments). (b) Modulated ΔpH between the supernatant and coacervate phase achieved through mixing of different PAAs with varying proportions of Z-Arg. (c) Time-dependent changes in pH of the supernatant and coacervate phases following the introduction of Na⁺, showing sustained proton extrusion (decrease in coacervate pH) coupled with Na⁺ uptake. Data are presented as mean ± SD (n = 3 independent experiments). (d) Macroscopic visualization showing the dynamic Na⁺–H⁺ exchange of SCPAAs: coacervates release protons upon NaCl introduction and subsequently uptake protons after phosphoric acid addition, demonstrating environmental responsiveness and proton-buffering capability.

While pH gradients across biomolecular condensates have recently been reportedparticularly in enzymatic condensates with more basic interiors, our system establishes a spontaneously sustained acidic core arising solely from nonequilibrium molecular self-assembly. Although direct measurement of ion fluxes was not pursued, a simple thermodynamic estimation indicates that the observed ΔpH (0.60–2.13) corresponds to a proton motive force of ≈3–12 kJ mol^–1 (ΔG = 2.303 RTΔpH at 25 °C), comparable to the free-energy span required for ion accumulation or for driving acid-catalyzed reactions under prebiotic conditions. Similar magnitudes of proton gradients have been proposed to enable protometabolic work in early Earth scenarios, , suggesting that the self-sustained pH differentials in SCPAAs could, in principle, support primitive chemiosmotic coupling even without protein machinery.

Strikingly, this proton gradient is not only stable but also readily tunable. By modulating the composition and ratios of the coacervate-forming library, we achieved programmable control of pH across a range of 0.6–4.0 (Figures b and S28), encompassing the acidic conditions (pH ∼ 1.5–5.0) geochemically plausible for early Earth environments such as volcanic hydrothermal systems. − For instance, coacervates formed from Z-Arg with Z-Pro, Z-Ile, or Z-Met maintained stable turbidity while showing reduced ΔpH, indicating compositional control over proton partitioning. Remarkably, increasing the proportion of Z-Arg reversed the gradient polarity, accompanied by the formation of Z-Arg–rich complex coacervates (Figure S29) in which the supernatant exhibited a lower pH than the coacervate interiora direct prebiotic analogue of bioenergetic gradients in modern cells. Such tunability highlights an intrinsic capacity for environmental adaptation, suggesting that these protocells could dynamically remodel their electrochemical identity to harness geochemically available energy sources.

Building on the spontaneously generated proton gradient that provides these coacervate protocells with a primitive energy currency, we examined whether such a pH differential could be harnessed to perform active worka defining hallmark of living systems. When exposed to elevated Na⁺ concentrations reminiscent of primordial seas, the SCPAAs indeed displayed facultative Na⁺–H⁺ antiport activity, dynamically exporting protons while importing Na⁺ to alleviate salinity stress and maintain ionic homeostasis. Direct quantification of the coacervate phase by inductively coupled plasma–optical emission spectrometry (ICP–OES) revealed a time-dependent increase in Na⁺ content, confirming Na⁺ influx (Figure S30a). Correspondingly, time-resolved pH measurements showed a gradual rise in coacervate pH accompanied by a decrease in supernatant pH within 24 h of salt addition (Figure c).

Thermogravimetric analysis indicated only a negligible (1.15%) loss of water over this period (Figure S30b, c), which would otherwise lower rather than raise the pH, thus excluding dehydration as the cause of the observed change and pinpointing Na⁺/H⁺ exchange as the operative mechanism. Remarkably, subsequent addition of phosphoric acid rapidly restored the initial low-pH state, demonstrating acute environmental responsiveness and a primitive proton-buffering capacity (Figures d and S31). Together, these findings show that SCPAAs can exploit their intrinsic proton gradient to drive active ion exchange, constituting a rudimentary form of chemiosmotic coupling and marking the emergence of energy transduction in a purely abiotic system.

Beyond establishing this minimal chemiosmotic behavior, such dynamic ion exchangerequiring no protein machineryeffectively places SCPAAs in a nonequilibrium steady state capable of harnessing electrochemical energy. This finding suggests that key bioenergetic processes, including chemiosmosis, could have arisen spontaneously from simple thermodynamic principles well before the advent of complex molecular transporters.

UV Radiation Tolerance

Ultraviolet (UV) radiation posed a major challenge to early life on Earth, damaging biomolecules in the absence of an ozone layer. , To assess whether SCPAA could mitigate this threat, we tested its ability to protect prebiotic molecules under simulated UV exposure. Glycine (Gly) encapsulated in SCPAA retained ∼86% of its initial concentration after 1 h of xenon lamp irradiation, compared to ∼84% in the unprotected control (Figure a). After 4 h, SCPAA-preserved Gly levels (∼34%) still exceeded controls (∼25%), demonstrating effective UV shielding.

5.

Exceptional tolerance and adaptability to harsh prebiotic conditions. (a) Variation of glycine relative content with UV irradiation time under the condition of with or without coacervates protection. Data are presented as mean ± SD (n = 3 independent experiments). (b) Protective effect of Z-Pro coacervates on different species exposed to UV for 1 h. Data are presented as mean ± SD (n = 3 independent experiments). (c) Fluorescent spectra and imaging of SCPAA with different excitation wavelengths, showing the multicolor emission of SCPAA. Scale bars: 100 μm. (d) Schematic representation illustrating the UV-resistant mechanism of SCPAA. (e) Pyruvic acid retention rates with and without SCPAA under varying heating durations. (f) Cooling and heating DSC curves of SCPAA. DSC cooling curve of SCPAA indicates water crystallization temperature. Heating curve reveals water melting temperature and SCPAA glass transition temperature (T _g). (g) Representative images of Z-Pro coacervates under increasing ionic stress, showing the morphological transition of coacervates from spread-out to more spherical geometries upon the addition of saturated NaCl solution or concentrated phosphoric acid (0 to 600 μL). (h) Quantitative analysis of coacervate morphological change. The aspect ratio (short-to-long axis ratio, %) of Z-Pro coacervates increases with the volume of added saturated NaCl solution or concentrated phosphoric acid, confirming a systematic shift toward a spherical morphology under high ionic strength.

To clarify the mechanism underlying this protection, we investigated whether the observed decrease in glycine concentration arose from molecular expulsion or photodecomposition. Expulsion was excluded, as the partition coefficient of glycine between the coacervate and supernatant phases increased following irradiation (Figure S32a), indicating no efflux occurred. Instead, the loss was attributed to UV-induced decomposition, supported by the detection of formamide and acetaldehydecharacteristic products of glycine decarboxylation and deaminationin the irradiated supernatant via ¹H NMR (Figure S32b). These results confirm that SCPAA protection arises from suppressing molecular decomposition rather than preventing diffusion losses.

Encapsulation also protected other aliphatic amino acids and peptides, with efficacy increasing in the order Gly₂ < Gly < Val (Figure b). This trend correlates with hydrophobicity (log P: Gly₂ = −1.85, Gly = −1.03, Val = 0.75), as hydrophobic molecules preferentially partition into SCPAA’s nonpolar microenvironment, enhancing their sequestration and protection. To assess the generality of this effect, we further examined the aromatic amino acid phenylalanine (Phe), which likewise exhibited significant protection (Figure S33). The slightly reduced protection relative to aliphatic amino acids is consistent with Phe’s strong intrinsic UV absorption (Figure b), conferring greater inherent photostability. Together, these results demonstrate that SCPAA provides a versatile photoprotective microenvironment, with protection magnitude modulated by molecular hydrophobicity and intrinsic stability.

The UV resilience of SCPAA is driven by three key mechanisms: (1) Physical barrier: Compartmentalization reduces direct UV exposure to encapsulated molecules; (2) UV absorption: SCPAA absorbs UV light via n→π* and π→π* transitions in its aromatic/hydrophobic motifs (Figure S34), shielding internalized cargo; (3) Red-edge excitation shift (REES): SCPAA exhibits wavelength-dependent fluorescence (Figure c), converting high-energy UV into lower-energy visible light (Figure S35). This spectral downshifting reduces photon energy reaching sensitive molecules. These synergistic effectscompartmentalization, absorption, and REEScollectively diminish UV-induced damage (Figure d). Crucially, SCPAA’s UV tolerance is generalizable across amino acids abundant in prebiotic environments, establishing it as a robust protective niche for primordial biomolecules. By preserving molecular integrity under harsh irradiation, SCPAA could have enabled the survival and evolution of primordial metabolic systems, bridging geochemistry and the emergence of life.

Resilience to Extreme Temperature Fluctuation

In addition to intense UV irradiation on primitive Earth, extreme temperature fluctuations characterized early terrestrial environments. These fluctuations would have compromised biomolecular integrity and metabolic activity, hindering life’s origin and evolution. To address this, we initially systematically analyzed the structural integrity of coacervate droplets under programmed temperature gradients. Phase-contrast microscopy analysis revealed a positive correlation between heating rates (2–50 K min^–1) and critical dissolution temperatures. Notably, droplets maintained structural integrity up to 70.0 °C even at a smaller heating rate of 2 K min^–1 (Figure S36).

We next quantified the protective function of SCPAA microcompartments using pyruvatea key energy source and biosynthetic precursor in primordial metabolismunder thermal stress (90.0 °C, 0–8 h). Chromogenic assays showed 60.4 ± 0.8% pyruvate preservation in SCPAA versus 29.8 ± 0.3% in aqueous controls (Figures e and S37). This protection correlates with persistent noncovalent interactions, evidenced by minimal changes in the Raman spectra of coacervates across heating durations (Figure S38). Such SCPAA systems could have provided stable microreactors for protometabolic cycles, reconciling destructive temperature swings with prebiotic chemistry preservation. Their thermal resilience supports the hypothesis that SCPAA compartments acted as evolutionary crucibles, driving chemical complexity under fluctuating conditions.

Beyond thermal stress, water crystallization at low temperatures threatens coacervate protocell stability through mechanical damage. To investigate this, we studied the low-temperature behavior of SCPAA using differential scanning calorimetry (DSC). The cooling DSC curve (Figure f) showed a water crystallization peak at −38.1 °C, confirmed by an ice melting peak at 0.4 °C upon heating. This result demonstrates SCPAA’s antifreezing property, where internal water molecules remain amorphous down to −38.1 °C. Crucially, a glass transition of the Z-Pro coacervate was detected at −43.5 °C. The close proximity between crystallization and glass transition temperature (ΔT = 5.4 °C) implies rapid vitrification mechanism that prevents ice-induced damage, analogous to the cryptobiotic strategies of extremophiles. This protective function is evidenced by the intact coacervate structure upon cooling from room temperature to −68.6 °C (Figure S39). Moreover, no birefringence was observed within SCPAA after crystallization, indicating that ice crystals were embedded within the glassy matrix and their growth was inhibited. These results are ascribed to the coexistence of “bound water” and “free water” within SCPAA, confirmed by low-field nuclear magnetic resonance relaxation spectra (Figure S40). While free water contributes to crystallization, tightly bound water acts as a plasticizer, depressing the T _g of Z-Pro SCPAA from −4.8 to −43.5 °C (Figure S41). This strong water-binding ability enhances coacervate resistance to low-temperature crystallization, potentially aiding early life survival.

Such behavior not only aligns with hypotheses of life’s origin in thermally variable niches but also challenges the conventional paradigm of membrane-bound protocells by offering an alternative pathway for maintaining compartmentalization under oscillating conditions. The observed thermal resilience, coupled with suppressed ice formation, positions this system as a robust platform for investigating prebiotic chemistry in astrobiologically relevant scenarios. Furthermore, its glass transition characteristics provide insights into preserving metabolic precursors during environmental stress.

Adaptive Reshaping under Prebiotic Ionic Stress

Having established their resilience to extreme temperature fluctuations, we next asked whether SCPAAs could also adapt to another pervasive feature of the prebiotic environmentdrastic oscillations in pH and salinity arising from geological and hydrological processes such as volcanic outgassing and hydrothermal circulation. To mimic these conditions, we introduced severe ionic perturbations into Z-Pro coacervates by adding either 85–90 wt % H₃PO₄ or saturated NaCl solution, simulating prebiotic ionic stress. Remarkably, rather than disassembling, the coacervates underwent a pronounced morphological transition from spread-out states to compact, spherical geometries (Figure g). This transition was quantified by monitoring the short-to-long axis ratio, which consistently approached unity with increasing ion concentration, confirming a shift toward energy-minimizing spheres under both highly acidic and high-salinity conditions (Figure h).

This morphological contraction was attributed to two synergistic factorscharge screening and dehydration-enhanced hydrophobic interactions. To verify this mechanism at the microphase level and rule out macroscopic artifacts, we next examined micron-scale coacervates, where surface tension effects dominate (Figure S42). These coacervate droplets maintained perfectly spherical morphologies yet exhibited a pronounced reduction in diameter under ionic stress (Figure 43), directly confirming enhanced molecular compaction. Together, these findings demonstrate that the adaptive reshaping originates from intrinsic physicochemical rearrangements within the coacervate phase rather than external deformation effects.

Such environment-dependent reshaping reveals a primitive yet efficient adaptive strategy that would have conferred selective advantages under prebiotic ionic stress. This dynamic morphological responsiveness not only reinforces the environmental robustness of SCPAAs but also suggests a potential route for prebiotic selection, whereby coacervates capable of preserving cohesion under fluctuating ionic conditions would have been preferentially retained, ultimately promoting the emergence of increasingly stable protocellular assemblies.

Evolutionary Potential of SCPAA toward Soft Membrane-like Structures

Beyond environmental resistance, an ideal coacervate protocell should evolve membrane-like structures at specific condition. Since simple polyanionic clusters form semipermeable membranes with organic cations, we tested whether membrane generation at the surface of SCPAA in the presence of trimetaphosphate, a prebiotically abundant compound. This design exploits electrostatic attraction between negatively charged trimetaphosphate and the positively charged SCPAA surface (Figure a). If formed, such polyanion-mediated membranes would be negatively charged, contrasting the positive coacervate core.

6.

Evolutionary potential of SCPAAs. (a) Schematic illustration demonstrating the charge distribution of the Z-Pro_mem coacervates surface. (b) Rhodamine B fluorescence in Z-Pro_mem coacervates. Peripheral fluorescence exceeded interior intensity, with interior fluorescence increasing over time. Scale bar: 100 μm. (c) Rhodamine B fluorescence in Z-Pro coacervates. Uniform intensity across droplets with no temporal change. Scale bar: 100 μm. (d) Fluorescence intensity profiles of Rhodamine B in Z-Pro_mem and Z-Pro coacervates. Z-Pro_mem coacervates showed peaks corresponding to membrane interfaces; Z-Pro coacervates exhibited no peaks. (e) Coalescence and sedimentation of Z-Pro_mem versus Z-Pro coacervates. Membrane-bearing Z-Pro_mem coacervates exhibited significantly slower coalescence and sedimentation rates than membrane-free Z-Pro counterparts. (f) Micrographs of Z-Pro_mem and Z-Pro coacervates. Z-Pro_mem coacervates resisted fusion at peripheries and within droplet interiors, whereas Z-Pro fused at both sites. Scale bars: 20 μm (Periphery); 100 μm (interior).

To detect membrane formation, cationic dye was selected as the probe. Adding Rhodamine B to Z-Pro coacervates with trimetaphosphate revealed significantly higher fluorescence intensity at the periphery versus the interior (Figure b, Left), indicating cationic dye accumulation at the surface. We term these coacervates Z-Pro_mem. Fluorescence within Z-Pro_mem coacervate droplets gradually increased over time (Figure b, Right), indicating slightly impeded dye diffusion into the core. Conversely, pristine Z-Pro droplets showed uniform fluorescence after dye addition, with rapid saturation and stabilization (Figure c). Fluorescence profiles indicated a ∼10 μm membrane thickness for Z-Pro_mem but no membrane peak for Z-Pro (Figure d). Critically, comparable membrane formation occurred in other SCPAA systems (Figure S44), demonstrating SCPAA’s evolutionary potential.

Having established membrane formation, we next examined its functional consequenceselective permeability. When exposed to the anionic dye indocyanine green (ICG), membrane-bearing Z-Promem coacervates displayed strongly reduced ICG uptake over 120 s, whereas membrane-free Z-Pro droplets were rapidly permeated (Figure S45, Videos S7, and S8). Together with the preferential enrichment of cationic Rhodamine B at the interface (Figure b), these results confirm that the trimetaphosphate-derived membrane introduces charge-selective permeabilitya hallmark property for maintaining distinct internal microenvironments.

Beyond molecular selectivity, the presence of a membrane also imparted pronounced structural stability. Membrane-bearing coacervates aggregated and sedimented much more slowly than their membrane-free counterparts (Figure e). Consistently, Z-Pro_mem droplets remained stably dispersed over extended periods, while Z-Pro droplets rapidly fused into larger aggregates (Figure f). Time-resolved measurements of droplet diameter further quantified this effect: Z-Pro coacervates exhibited rapid growth due to fusion, whereas Z-Pro_mem maintained a nearly constant size over time, demonstrating long-term suppression of coalescence (Figure S46). This kinetic stabilization preserves protocell individuality, enabling persistent compartmentalization of internal reactions such as replication or catalysis. By maintaining structural integrity and distinct internal environments, the membranized coacervates fulfill a key prerequisite for evolutionary processesallowing selection to act on individual entities. Moreover, this acquired stability and individuality represent an essential step toward more complex behaviors, such as division and reproduction, thereby bridging the gap between simple prebiotic assemblies and cell-like systems.

Conclusions

In summary, we have shown that prebiotically plausible amino acid derivatives, incorporating hydrophobic motifs and flexible spacers, self-assemble through liquid–liquid phase separation into membraneless coacervate protocells (SCPAAs). These dynamic assemblies are stabilized by hydrophobic interactions and water-mediated noncovalent networks, and exhibit three key functionalities essential for early protocell models: they provide an unique microenvironment to enhance enzyme-free metabolic reactions such as sulfur redox chemistry and prebiotic pigment synthesis; they display remarkable resistance to environmental stressors, including high salinity, divalent cations, UV radiation, extreme temperatures, and pH fluctuations, while adaptively remodeling under ionic stress; and most significantly, they autonomously generate and maintain proton gradients across their interfaces, enabling primitive chemiosmotic coupling via Na⁺–H⁺ antiport.

By unifying compartmentalization, nonenzymatic catalysis, energy transduction, and environmental resilience within a geochemically relevant system constructed from simple amino acid building blocks, SCPAAs provide a compelling link between prebiotic chemistry and the emergence of cellular life. Unlike membrane-bound or continuously fueled models, these structures operate robustly under prebiotically plausible conditions through self-organization and nonequilibrium thermodynamics. This work establishes a minimalist yet multifunctional protocell platform that illustrates how primitive compartments could have sustained metabolic complexity, maintained ion gradients, and undergone physical selection prior to the evolution of lipid membranes. Our findings offer a unifying framework for understanding the chemical and thermodynamic principles underlying the origin of evolvable protocells on the early Earth.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c15328.

• Materials; methods; thermal stability of Boc-Gly and Boc-Pro; chemical structures of Z-amino acids and Boc-amino acids; list of melting point, Log P, and coacervates formation ability of Z-amino acids and Boc-amino acids; bright field images; fluorescent images; dynamic light scattering intensity profiles; macroscopic images; bright-field optical images; thermodynamic calculation parameters; changes in water content; optimized cluster configuration; metal ions-encapsulated Z-Pro and Boc-Ala coacervates; Raman spectra of Z-Pro coacervates and Boc-Ala; UV-vis spectra of SCPAA; low-resolution ESI-MS spectrum; time-dependent yield of cystine; concentration of L-Cys; formation of melanin-like derivatives; time-dependent UV-vis spectra; FRAP recovery curves; sedimentation of Z-Tyr_mel versus Z-Tyr coacervates; morphology of Z-Tyr coacervates; time-dependent curves of pH; pH of the supernatant and coacervate; mass spectra of the coacervate system; quantification determination; visualization of the pH gradient; size changes; comparative time-lapse images; partition coefficients; UV-protecting effect of Z-Pro coacervates; UV-vis spectrum; correlation between the excitation and emission peak wavelengths; retention of Z-Pro coacervate droplets; mechanism of pyruvic acid detection; distinct hydration states; and size changes of Z-Pro coacervate droplets and Z-Pro_mem coacervate droplets (PDF)

• Videos-Part 1: cooling; 3D confocal image of Z-pro coacervates; coacervating, fusing, and deforming (ZIP)

• Videos-Part 2: the formation process of cystine crystallization in the presence of SCPAA; cystine crystallization did not occur within 2 hr in water; ICG enters into Z-Pro_mem coacervates and Z-Pro coacervates (ZIP)

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S.C. and G.L. contributed equally to this work.

The authors declare no competing financial interest.